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The Similarity of Action Spectra for Thymine Dimers in Human Epidermis and Erythema Suggests that DNA is the Chromophore for Erythema

1998; Elsevier BV; Volume: 111; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.1998.00436.x

1523-1747

Antony R. Young, Graham I. Harrison, Caroline Chadwick, Osamu Nikaido, J. Ramsden, Christopher S. Potten,

Immunotherapy and Immune Responses

The location of DNA photodamage within the epidermis is crucial as basal layer cells are the most likely to have carcinogenic potential. We have determined the action spectra for DNA photodamage in different human epidermal layersin situ. Previously unexposed buttock skin was irradiated with 0.5, 1, 2, and 3 minimal erythema doses of monochromatic UVR at 280, 290, 300, 310, 320, 340, and 360 nm. Punch biopsies were taken immediately after exposure and paraffin sections were prepared for immunoperoxidase staining with a monoclonal antibody against thymine dimers that were quantitated by image analysis. Dimers were measured at two basal layer regions, the mid and the upper living epidermis. The slopes of dose–response curves were used to generate four action spectra, all of which had maxima at 300 nm. Dimer action spectra between 300 and 360 nm were independent of epidermal layer, indicating comparable epidermal transmission at these wavelengths. Furthermore, we observed 300 nm-induced dimers in dermal nuclei; however, there was a marked effect of epidermal layer between 280 and 300 nm, showing relatively poor transmission of 280 and 290 nm to the basal layer. These data indicate that solar UVB (≈295–320 nm) is more damaging to basal cells than predicted from transmission data obtained from human epidermisex vivo. The epidermal dimer action spectra were compared with erythema action spectra determined from the same volunteers and ultraviolet radiation sources. Overall, these spectral comparisons suggest that DNA is a major chromophore for erythema in the 280–340 nm region. The location of DNA photodamage within the epidermis is crucial as basal layer cells are the most likely to have carcinogenic potential. We have determined the action spectra for DNA photodamage in different human epidermal layersin situ. Previously unexposed buttock skin was irradiated with 0.5, 1, 2, and 3 minimal erythema doses of monochromatic UVR at 280, 290, 300, 310, 320, 340, and 360 nm. Punch biopsies were taken immediately after exposure and paraffin sections were prepared for immunoperoxidase staining with a monoclonal antibody against thymine dimers that were quantitated by image analysis. Dimers were measured at two basal layer regions, the mid and the upper living epidermis. The slopes of dose–response curves were used to generate four action spectra, all of which had maxima at 300 nm. Dimer action spectra between 300 and 360 nm were independent of epidermal layer, indicating comparable epidermal transmission at these wavelengths. Furthermore, we observed 300 nm-induced dimers in dermal nuclei; however, there was a marked effect of epidermal layer between 280 and 300 nm, showing relatively poor transmission of 280 and 290 nm to the basal layer. These data indicate that solar UVB (≈295–320 nm) is more damaging to basal cells than predicted from transmission data obtained from human epidermisex vivo. The epidermal dimer action spectra were compared with erythema action spectra determined from the same volunteers and ultraviolet radiation sources. Overall, these spectral comparisons suggest that DNA is a major chromophore for erythema in the 280–340 nm region. cyclobutane pyrimidine dimer just perceptible minimal erythema dose mean optical density thymine dimer Solar ultraviolet radiation (UVR) causes skin cancer (Green and Williams, 1993Green A. Williams G. Ultraviolet radiation and skin cancer: Epidemiological data from Australia.in: Young A.R. Bjorn L.O. Moan J. Nultsch W. Environmental UV Photobiology. Plenum Press, New York1993: 233-254Google Scholar;de Gruijl and van der Leun, 1994de Gruijl F.R. van der Leun J.C. Estimate of the wavelength dependency of ultraviolet carcinogenesis in humans and its relevance to the risk assessment of a stratospheric ozone depletion.Health Physics. 1994; 67: 1-8Crossref Scopus (198) Google Scholar), especially in sun-sensitive people (skin types I/II) who tan poorly. DNA is a major epidermal chromophore (Young, 1997Young A.R. Chromophores in human skin.Physics Med Biol. 1997; 42: 789-802Crossref PubMed Scopus (229) Google Scholar) and there is increasing evidence that DNA photodamage, such as cyclobutane pyrimidine dimers (CPD) and consequent mutation (e.g., p53), have a direct role in the initiation of skin tumors (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1302) Google Scholar). Mouse studies provide evidence that UVR-induced immunosuppression, which specifically inhibits the normal immunosurveillance of UVR-induced tumor cells, may be mediated via epidermal CPD (Kripke et al., 1992Kripke M.L. Cox P.A. Alas L.G. Yarosh D.B. Pyrimidine dimers in DNA initiate systemic immunosuppression in UV-irradiated mice.Proc Natl Acad Sci USA. 1992; 89: 7516-7520Crossref PubMed Scopus (442) Google Scholar). Overall, DNA is suspected of being a chromophore for many of the photobiologic effects of UVR. These include cytokine induction (Yarosh and Kripke, 1996Yarosh D.B. Kripke M.L. DNA repair and cytokines in antimutagenesis and anticarcinogenesis.Mutation Res. 1996; 350: 255-260Crossref PubMed Scopus (25) Google Scholar), which is likely to be important in UVR-induced immunosuppression and acute inflammation (erythema/sunburn) (Ley, 1985Ley R.D. Photoreactivation of UV-induced pyrimidine dimers and erythema in the marsupialMonodelphis domestica.Proc Natl Acad Sci USA. 1985; 82: 2409-2411Crossref PubMed Scopus (97) Google Scholar), and tanning (Gilchrest et al., 1996Gilchrest B.A. Park H.-Y. Eller M.S. Yaar M. Mechanisms of ultraviolet light-induced pigmentation.Photochem Photobiol. 1996; 63: 1-10Crossref PubMed Scopus (299) Google Scholar). The location of DNA photolesions within the epidermis is likely to be very important in tumor initiation. Mutation to suprabasal keratinocytes committed to terminal differentiation is likely to be inconsequential, whereas DNA damage and mutation to stem cells in the basal layer may have serious long-term consequences. Most studies of UVR-induced DNA damage in human skin have been based on techniques that require the digestion of the epidermis (Freeman et al., 1989Freeman S.E. Hacham H. Gange R.W. Maytum D.J. Sutherland J.C. Sutherland B.M. Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiatedin situ with ultraviolet light.Proc Natl Acad Sci USA. 1989; 86: 5605-5609Crossref PubMed Scopus (252) Google Scholar;Bykov and Hemminki, 1996Bykov V.J. Hemminki K. Assay of different photoproducts after UVA, B and C irradiation of DNA and human skin explants.Carcinogenesis. 1996; 17: 1949-1955Crossref PubMed Scopus (21) Google Scholar) prior to DNA extraction, thereby preventing the localization of damage. We have recently developed monoclonal antibody-based image analysis techniques to quantitate DNA photodamage in human epidermisin situ (Potten et al., 1993Potten C.S. Chadwick C.A. Cohen A.J. Nikaido O. Matsunaga T. Schipper N.W. Young A.R. DNA damage in UV-irradiated human skinin vivo: automated direct measurement by image analysis (thymine dimers) compared with indirect measurement (unscheduled DNA synthesis) and protection by 5-methoxypsoralen.Int J Radiation Biol. 1993; 63: 313-324Crossref PubMed Scopus (50) Google Scholar;Young et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten S. Thein situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 307-1313Google Scholar). A major advantage of this approach is that specific DNA lesions and cell types can be localized within the epidermis (Chadwick et al., 1995Chadwick C.A. Potten C.S. Nikaido O. Matsunaga T. Proby C. Young A.R. The detection of cyclobutane thymine dimers, (6–4) photolesions and the Dewar photoisomers in sections of UV-irradiated human skin using specific antibodies, and the demonstration of depth penetration effects.J Photochem Photobiol B. Biol. 1995; 28: 163-170Crossref PubMed Scopus (101) Google Scholar;Young et al., 1998Young A.R. Potten C.S. Nikaido O. Parsons P.G. Boenders J. Ramsden J.M. Chadwick C.A. Human melanocytes and keratinocytes exposed to UVB or UVAin vivo show comparable levels of thymine dimers.J Invest Dermatol. 1998; 111: 936-940Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). We have exploited this advantage to determine action spectra for thymine dimers (TT), a specific type of CPD, in different layers of human epidermis. In addition, we have determined the action spectra for erythema, with qualitative and quantitative techniques, in the same group of volunteers. An action spectrum, which demonstrates the relative photobiologic efficiency at different wavelengthsper se, can be used to identify chromophores and/or as a biologic weighting function to calculate the relative importance of different wavelengths under actual environmental conditions. The latter could, for example, evaluate the effects of ozone layer depletion on basal layer DNA photodamage. Whereas several human erythema action spectra have been published (Parrish et al., 1982Parrish J.A. Jaenicke K.F. Anderson R.R. Erythema and melanogenesis action spectra of normal human skin.Photochem Photobiol. 1982; 36: 187-191Crossref PubMed Scopus (366) Google Scholar;McKinlay and Diffey, 1987McKinlay A.F. Diffey B.L. A reference action spectrum for ultraviolet induced erythema in human skin.Cie J. 1987; 6: 17-22Google Scholar;Anders et al., 1995Anders A. Altheide H.-J. Knalmann M. Knälmann M. Tronnier H. Action spectrum for erythema in humans investigated with dye lasers.Photochem Photobiol. 1995; 61: 200-205Crossref PubMed Scopus (78) Google Scholar), our specific aim was to evaluate the significance of epidermal DNA as a chromophore for erythema by comparing TT and erythema action spectra in the same volunteer pool with the same irradiation sources and protocols. Apart from the mechanistic information that such a comparison might provide, we were interested in the value of erythema as a noninvasive clinical surrogate for DNA photodamage. Forty skin type I/II volunteers (healthy young adults) were recruited for studies at 280, 290, 300, 310, 320, 340, and 360 nm (n = 6 for each wavelength except at 310 nm with n = 4). Demographic analyses are shown inTable 1. The study was approved by the Ethics Committee of St. Thomas' Hospital (London, U.K.), and written informed consent was obtained from all participants.Table 1Source attributes, demographic analyses, and median MED at different wavelengthsaNote that at 320, 340, and 360 nm WG320 1 mm filters were used to reduce stray UVB radiation. In two cases skin types were defined as type I/II as we could not fit them into either category. The age range of volunteers was restricted to exclude any possible effects of aging. The mean age was 25.8 ± 5.7 (SD). Note that bandwidth refers to full-width half-maximum, which is the bandwidth at which 50% maximal irradiance occurs. Median rather than mean MED is given because MED has been reported to be log-normally distributed in a normal population ( Mackenzie 1983).SexSkin typeλ (nm)Bandwidth (nm)MFIIII/IIMedian age (y)Median MED (J per cm2)2805151520.00.05290515628.50.035300515629.00.0253105221324.00.25320105132124.02.43402051625.524.036020335125.032.0All λ1822533225.5a Note that at 320, 340, and 360 nm WG320 1 mm filters were used to reduce stray UVB radiation. In two cases skin types were defined as type I/II as we could not fit them into either category. The age range of volunteers was restricted to exclude any possible effects of aging. The mean age was 25.8 ± 5.7 (SD). Note that bandwidth refers to full-width half-maximum, which is the bandwidth at which 50% maximal irradiance occurs. Median rather than mean MED is given because MED has been reported to be log-normally distributed in a normal population ( Mackenzie, 1983Mackenzie L.A. The analysis of ultraviolet radiation doses required to produce erythema responses in normal skin.Br J Dermatol. 1983; 108: 1-9Crossref PubMed Scopus (25) Google Scholar). Open table in a new tab Monochromatic spectra were obtained from a Photo-irradiation System (Applied Photophysics, London, U.K.) with a 2 kW xenon arc. Delivery of UVR was with a liquid light guide (Oriel, Leatherhead, U.K.) with a 5 mm diameter exit. Irradiance was routinely measured with a wide-band thermopile radiometer designed to accommodate the light guide (Medical Physics, Dryburn Hospital, Durham, U.K.). Irradiances increased with wavelength, ranging from about 1.0–1.5 mW per cm2 at ≤ 300 nm to about 50 mW per cm2 at 360 nm. Source attributes are shown inTable 1, and their normalized emission spectra, determined with a Bentham DM 150 double monochromator spectroradiometer (Bentham Instruments, Reading, U.K.) calibrated with a deuterium source measured by the National Physics Laboratory (Teddington, U.K.), are shown inFigure 1. A perspex applicator with six apertures, designed to accommodate the exit end of the liquid light guide, was attached with adhesive tape to a flat area of previously unexposed buttock skin. After each exposure the light guide was moved to the next aperture. Each person was exposed to an assigned wavelength and also to 300 nm, which was used as a reference for comparing the responses of the different wavelength groups. At each waveband, a geometric series of six exposures was given with a dose increment of √2. Dose ranges were determined in pilot studies. Twenty-four hours after exposure, erythema intensity at each site was assessed visually by at least two observers according to the following scale: 0, no reaction or observers not sure; 0.5, minimal perceptible erythema (no clear borders and defined as the MED); 1.0, definite erythema with clear borders; 2, erythema with edema; and 3, edema or blisters. In addition, there was triplicate quantitative assessment of erythema intensity (arbitrary units) at each site, as well as an adjacent nonirradiated control site to provide a background erythema level, made with a PC-linked Dia-Stron erythema (reflectance) meter (Dia-Stron, Andover, U.K.). Individual MED was used as the fundamental exposure unit for the subsequent dose–response studies for TT. For these, irradiation procedures were as before, on the contralateral buttock, and each person was exposed to 0.5, 1.0, 2.0, and 3.0 MED. The maximum time taken to deliver 1 MED ranged from about 15–30 s with wavelengths ≤ 300 nm to about 15 min at 360 nm. Punch biopsies (4 mm) were taken from each site, under local anesthesia, immediately after exposure (in practice within 5 min) as well as a control biopsy from an adjacent nonexposed site. The details have been published elsewhere (Potten et al., 1993Potten C.S. Chadwick C.A. Cohen A.J. Nikaido O. Matsunaga T. Schipper N.W. Young A.R. DNA damage in UV-irradiated human skinin vivo: automated direct measurement by image analysis (thymine dimers) compared with indirect measurement (unscheduled DNA synthesis) and protection by 5-methoxypsoralen.Int J Radiation Biol. 1993; 63: 313-324Crossref PubMed Scopus (50) Google Scholar;Chadwick et al., 1995Chadwick C.A. Potten C.S. Nikaido O. Matsunaga T. Proby C. Young A.R. The detection of cyclobutane thymine dimers, (6–4) photolesions and the Dewar photoisomers in sections of UV-irradiated human skin using specific antibodies, and the demonstration of depth penetration effects.J Photochem Photobiol B. Biol. 1995; 28: 163-170Crossref PubMed Scopus (101) Google Scholar;Young et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten S. Thein situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 307-1313Google Scholar). Briefly, within 5 min, the biopsies were sliced into 1 mm strips and fixed in methanol at 4°C for 18 h, from which 3 μm sections were cut. These were immunostained with a monoclonal antibody for TT (TDM-1) (Mizuno et al., 1991Mizuno T. Matsunaga T. Ihara M. Nikaido O. Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA. a comparative study with 64M–1 antibody specific for (6–4) photoproducts.Mutation Res. 1991; 254: 175-184Crossref PubMed Scopus (88) Google Scholar) followed by a DAB-peroxidase immune reaction that gives brown nuclear coloration. Nuclei were counter-stained with thionine. The sections were analyzed using the Discovery automated image analysis system (Becton Dickinson, Leiden, The Netherlands) in which the nuclei are identified and defined with the thionine blue stain using a 620 nm filter and the antibody reaction product is quantitated using a 460 nm filter, within the defined nuclear mask, and expressed as a mean optical density (MOD) per nucleus. Standard sections were always included in each staining run to verify staining, i.e., MOD, reproducibility. The intensity of the light was always set at a constant level and the MOD of a reference sample was confirmed to insure that intergroup comparisons could be made. Four epidermal layers were defined by an interactive process with the image analysis software: (i) upper, the outermost two nucleated layers; (ii) mid, the next two cell layers down; (iii) basal 5–7, basal cells at a depth of 5–7 cells; and (iv) basal 8+, basal cells at a depth of eight or more layers. Two assessments were made in the basal layer to accommodate its undulating nature. At least 150 nuclei were assessed per cell layer per skin section. In each volunteer, the specific background MOD (i.e., zero MED) was subtracted from the MOD of the UVR exposed sites/layers. In all cases, the individual biologic dose units (MED) were converted to physical units (J per cm2). Using data generated from the reflectance meter, the quantitative measurement of erythema was determined by the difference (ΔE) between the reading from a UVR-exposed site and the background value from a nonexposed site. UVR dose–response curves were constructed as a logit function of log10 UVR dose according toDiffey and Farr, 1991Diffey B.L. Farr P.M. Quantitative aspects of ultraviolet erythema.Clin Physics Physiol Measurement. 1991; 12: 311-325Crossref PubMed Scopus (70) Google Scholar. The dose to achieve ΔE = 50 (DΔE, equivalent to ≈1 MED assessed by eye) was calculated from the logit regression parameters. Erythema action spectra were determined with qualitative and quantitative data using the classic log101/MED endpoint and log101/DΔE, respectively. Linear regression analysis was used to determine UVR dose–response curves for TT at each wavelength and each epidermal layer for each volunteer. It was considered best to fit all data to the same model and linear regression was chosen as the simplest. Individual data were pooled to generate mean log10 slope for TT induction ± SD for each wavelength at each epidermal location. Action spectra for TT induction were generated by plotting mean log10 slope against wavelength. The median 300 nm MED for the whole volunteer group (n = 39, one not done) was 0.032 J per cm2. The values at 300 nm for the groups also exposed to 280, 290, 310, 320, 340, and 360 nm were 0.030, 0.035, 0.032, 0.028, 0.035, and 0.040 J per cm2, respectively, with a ratio of 1.6 between the groups with the highest (360 nm) and the lowest (300 nm, seeTable 1) MED. This is about 14% more than a single exposure-dose increment (i.e., ×1.4), indicating that all groups showed a comparable erythema response at 300 nm. Figure 2 shows that erythema action spectra determined by clinical assessment of MED and by analysis of reflectance data are the same. The quality of reflectance data at 280 nm was not as good as with other wavelengths, i.e., increased dose did not always result in increased erythema. We suspect that this is due to local variations in stratum corneum thickness. Typical individual linear regression analysis of TT dose–response for each wavelength are shown inFigure 3. In general, the regression fits were good withR2 ≥ 0.85 in 32% cases, ≥ 0.75 in 22% cases, and ≥ 0.65 in 10% cases. The data points represent the individualised doses of 0.5, 1, 2, and 3 MED. They show that there is a clear epidermal layer effect on the dose–response curves at 280 and 290 nm with less steep slopes in the deeper layers. It should be noted that the quality of the data at 280 and 290 nm was less good than at other wavelengths, probably because of local variations in stratum corneum thickness.Figure 4a) shows histologic evidence for the lack of epidermal penetration at 290 nm; however, at wavelengths ≥ 300 nm the dose–response curves for all epidermal layers can be superimposed, even at the lower doses. This clearly demonstrates that saturation of the response has not occurred and that TT are induced by suberythemal exposures at all wavelengths.Figure 4b,c shows histologic evidence for the lack of epidermal screening at 300 nm as well as the presence of TT in dermal cells. The action spectra for TT in the four different epidermal layers are shown inFigure 5. This overall analysis, based on the slopes of the regression curves, shows that between 300 and 360 nm, the action spectra are independent of epidermal depth; however, epidermal location modifies the spectra between 280 and 300. The shorter UVB wavelengths are much less effective at damaging the deep basal cells indicating decreased epidermal transmission. A comparison between the erythema and TT action spectra is shown inFigure 6. Between 280 and 340 nm, the erythema action spectrum shows excellent concordance with the TT action spectra of the upper and mid epidermis, but deviation from the basal layers. All spectra show excellent concordance between 300 and 340 nm. At 360 nm there is evidence of deviation between the erythema and TT action spectra. Figure 7 shows the relationship between our TT epidermal action spectra (quantum corrected) and the quantum corrected TT action spectrum in naked DNA using the same antibody (Matsunaga et al., 1991Matsunaga T. Hieda K. Nikaido O. Wavelength dependent formation of thymine dimers and (6–4) photoproducts in DNA by monochromatic ultraviolet light ranging from 150 to 365 nm.Photochem Photobiol. 1991; 54: 403-410Crossref PubMed Scopus (173) Google Scholar). The latter, as expected, shows a good match with the DNA absorption spectrum (Sutherland and Griffin, 1981Sutherland J.C. Griffin K.P. Absorption spectrum of DNA for wavelengths greater than 300 nm.Radiation Res. 1981; 86: 399-409Crossref PubMed Scopus (155) Google Scholar). There is a goodin vivo/in vitro match of TT action spectra between 300 and 320 nm but the lack of match at wavelengths less than 300 nm demonstrates the marked screening effect of epidermal chromophores. At 320–360 nm higher doses are required for TT inductionin vivo than would be expected from thein vitro TT data. We have determined action spectra for TT induction in different layers of human epidermisin vivo. Our data show that 300 nm is the most effective wavelength in the solar UVR range, irrespective of epidermal layer. Maximal efficacy in human epidermis at 300 nm was also reported byFreeman et al., 1989Freeman S.E. Hacham H. Gange R.W. Maytum D.J. Sutherland J.C. Sutherland B.M. Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiatedin situ with ultraviolet light.Proc Natl Acad Sci USA. 1989; 86: 5605-5609Crossref PubMed Scopus (252) Google Scholar in a study using the endonuclease sensitive site technique to determine CPD levels in whole human epidermis. A mouse study assessing epidermal CPD showed a peak at 293 nm (Ley et al., 1983Ley R.D. Peak M.J. Lyon L.L. Induction of pyrimidine dimers in epidermal DNA of hairless mice by UVB. an action spectrum.J Invest Dermatol. 1983; 80: 188-191Abstract Full Text PDF PubMed Scopus (41) Google Scholar). TT action spectra were dependent on epidermal depth in the 280–300 nm range, with 280 nm causing almost 40-fold less damage (slope comparisons) to the deep basal layer than the upper layer. DNA damage, however, was independent of epidermal layer at wavelengths between 300 and 360 nm, clearly demonstrating a lack of screening, and therefore damage gradient, within the epidermis in this spectral region. We have previously reported a lack of a gradient within human epidermis for TT induced by 300 nm but a steep gradient was seen at 260 nm (Chadwick et al., 1995Chadwick C.A. Potten C.S. Nikaido O. Matsunaga T. Proby C. Young A.R. The detection of cyclobutane thymine dimers, (6–4) photolesions and the Dewar photoisomers in sections of UV-irradiated human skin using specific antibodies, and the demonstration of depth penetration effects.J Photochem Photobiol B. Biol. 1995; 28: 163-170Crossref PubMed Scopus (101) Google Scholar). These observations are confirmed in this study, in which a gradient was seen at 280 nm and 290 nm but not with longer wavelengths. A similar lack of gradient was also observed for 300 nm-induced epidermal p53 expression, although a gradient was seen with 254 nm (Campbell et al., 1993Campbell C. Quinn A.G. Angus B. Farr P.M. Rees J.L. Wavelength specific patterns of p53 induction in human skin following exposure to UV radiation.Cancer Res. 1993; 53: 2697-2699PubMed Google Scholar).Bykov and Hemminki, 1996Bykov V.J. Hemminki K. Assay of different photoproducts after UVA, B and C irradiation of DNA and human skin explants.Carcinogenesis. 1996; 17: 1949-1955Crossref PubMed Scopus (21) Google Scholar compared CPD levels in naked DNA and human epidermis induced by 0.02 J UVB and UVC per cm2 (similar to our MED). Although UVC-induced CPD levels in epidermis were lower than naked DNA, a greater number of UVB-induced CPD/nucleotide was observed in the epidermis. Overall, our data and those of others show that UVR transmission properties determinedin vitro from human epidermal sheets (Bruls et al., 1984Bruls W.A. Slaper H. van der Leun J.C. Berens L. Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths.Photochem Photobiol. 1984; 40: 485-494Crossref PubMed Scopus (390) Google Scholar) underestimate UVB-induced basal layer damage and may not be relevant to human skinin vivo. Furthermore, our data suggest that the use ofin vitro transmission data to correct mouse action spectra for nonmelanoma skin cancer for human risk assessment (de Gruijl and van der Leun, 1994de Gruijl F.R. van der Leun J.C. Estimate of the wavelength dependency of ultraviolet carcinogenesis in humans and its relevance to the risk assessment of a stratospheric ozone depletion.Health Physics. 1994; 67: 1-8Crossref Scopus (198) Google Scholar), may underestimate the potential carcinogenic effects of ozone layer depletion. As expected from our data, visual inspection of the skin sections shows evidence of UVB (300 nm)-induced DNA photolesions in dermal cells, although this has not been quantitated nor have the cells been identified; however, we believe that it is likely that solar UVB may have a direct effect of the skin's vasculature, especially in the papillary region. DNA shows an absorption maximum at about 260 nm with an absorption tail in the UVB region (Sutherland and Griffin, 1981Sutherland J.C. Griffin K.P. Absorption spectrum of DNA for wavelengths greater than 300 nm.Radiation Res. 1981; 86: 399-409Crossref PubMed Scopus (155) Google Scholar). Thein vitro action spectrum for TT, with the same antibody as ourin vivo study, is generally similar in shape to the DNA absorption spectrum with a maximum at 260 nm (Matsunaga et al., 1991Matsunaga T. Hieda K. Nikaido O. Wavelength dependent formation of thymine dimers and (6–4) photoproducts in DNA by monochromatic ultraviolet light ranging from 150 to 365 nm.Photochem Photobiol. 1991; 54: 403-410Crossref PubMed Scopus (173) Google Scholar). At wavelengths less than 300 nm, the differences between the epidermal TT action spectra and the DNA absorption spectrum or the TTin vitro action spectrum, are likely to be accounted for by the optical screening properties of the epidermis. Differences in the optical properties of mouse and human epidermis, especially the stratum corneum, may account for the mouse CPD action spectrum maximum at 293 nm (Ley et al., 1983Ley R.D. Peak M.J. Lyon L.L. Induction of pyrimidine dimers in epidermal DNA of hairless mice by UVB. an action spectrum.J Invest Dermatol. 1983; 80: 188-191Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Our data show that the living part of the epidermis has very little effect on UVR transmission in the 300–360 nm range, but attenuates wavelengths less than 300 nm. The 300 nm peak in the TT action spectrum of the upper living epidermis, although less pronounced than the lower layers, provides evidence of considerable screening of the shorter wavelengths (i.e., < 300 nm) by chromophores in the nonviable epidermis. Urocanic acid, which is found in uniquely high concentrations (6–12 nmole per cm2) in the stratum corneum, is the most likely candidate because of its peak absorption at about 268 nm and, like DNA, its absorption tail in the UVB region. Our data show that, in the 320–360 nm region, higher doses are required for TT inductionin vivo than would be expected fromin vitro data. We cannot explain this but it is possible that our data provide indirect evidence for UVA-induced enzymatic photoreactivation (namely TT monomerization), which has been reported to occur within 30 min (Sutherland et al., 1992Sutherland B.M. Hacham H. Gange R.W. Sutherland J.C. Pyrimidine dimer formation by UVA radiation: Implications for photoreactivation.in: Urbach F. Biological Responses to Ultraviolet a Radiation. Valdenmar Publishing, Kansas1992: 47-58Google Scholar) although the role of this process in human skin remains controversial. We have demonstrated the presence of the 6–4 photoproduct in human skinin vivo after irradiation with solar simulated radiation and with 260 nm (Chadwick et al., 1995Chadwick C.A. Potten C.S. Nikaido O. Matsunaga T. Proby C. Young A.R. The detection of cyclobutane thymine dimers, (6–4) photolesions and the Dewar photoisomers in sections of UV-irradiated human skin using specific antibodies, and the demonstration of depth penetration effects.J Photochem Photobiol B. Biol. 1995; 28: 163-170Crossref PubMed Scopus (101) Google Scholar;Young et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten S. Thein situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 307-1313Google Scholar). The action spectrum for the 6–4 photoproduct in human skin is not known but, based onin vitro data (Rosenstein and Mitchell, 1987Rosenstein B.S. Mitchell D.L. Action spectra for the induction of pyrimidine (6–4) pyrimidone photoproducts and cyclobutane pyrimidine dimers in normal human skin fibroblasts.Photochem Photobiol. 1987; 45: 775-778Crossref PubMed Scopus (151) Google Scholar;Matsunaga et al., 1991Matsunaga T. Hieda K. Nikaido O. Wavelength dependent formation of thymine dimers and (6–4) photoproducts in DNA by monochromatic ultraviolet light ranging from 150 to 365 nm.Photochem Photobiol. 1991; 54: 403-410Crossref PubMed Scopus (173) Google Scholar), it is reasonable to assume that it is essentially similar to that for TT in the solar UVB region. Erythema action spectra determined by visual assessment or by analysis of dose–response data determined by reflectance were very similar, confirming the value of the MED concept. These action spectra were similar, in qualitative and quantitative terms, to those published by others (Parrish et al., 1982Parrish J.A. Jaenicke K.F. Anderson R.R. Erythema and melanogenesis action spectra of normal human skin.Photochem Photobiol. 1982; 36: 187-191Crossref PubMed Scopus (366) Google Scholar;McKinlay and Diffey, 1987McKinlay A.F. Diffey B.L. A reference action spectrum for ultraviolet induced erythema in human skin.Cie J. 1987; 6: 17-22Google Scholar) and, for example, to a recent study, based on 252 normal volunteers of skin types I, II, and III, which reported a median 300 nm MED of 0.027 J per cm2 with a 95% range of 0.015–0.051 J per m2 (Diffey, 1994Diffey B.L. Observed and predicted minimal erythema doses: a comparative study.Photochem Photobiol. 1994; 60: 380-382Crossref PubMed Scopus (23) Google Scholar). This is close to our median 300 nm MED of 0.032 J per cm2. A recent human erythema action spectrum shows a steeper decline in the 300–320 nm region (Anders et al., 1995Anders A. Altheide H.-J. Knalmann M. Knälmann M. Tronnier H. Action spectrum for erythema in humans investigated with dye lasers.Photochem Photobiol. 1995; 61: 200-205Crossref PubMed Scopus (78) Google Scholar) than earlier studies. The reason for this is almost certainly the use of true monochromatic radiation from a laser source, which prevents the "contaminating" effects of wavelengths lower than peak emission that are present in spectra from conventional monochromators. There was a striking concordance between the erythema and the mid and upper layer TT action spectra between 280 and 340 nm. This agreement was independent of cell layer between 300 and 340 nm. Our endpoint for erythema was based on a threshold response, whether assessed by eye or machine. It has long been established that erythema dose–response curves become less steep with wavelengths decreasing from UVB towards UVC (reviewed byDiffey and Farr, 1991Diffey B.L. Farr P.M. Quantitative aspects of ultraviolet erythema.Clin Physics Physiol Measurement. 1991; 12: 311-325Crossref PubMed Scopus (70) Google Scholar) and confirmed by our 280 and 290 nm erythema dose–response data (not shown). An important consequence of this is that the shape of the action spectrum for erythema, at wavelengths less than 300 nm, depends on the degree of erythema that is used as the endpoint. Relative to 300–310 nm, a much higher dose of shorter wavelengths is necessary to induce an intense erythema. Thus, the calculated action spectrum for an intense erythema (Farr and Diffey, 1985Farr P.M. Diffey B.L. The erythemal response of human skin to ultraviolet radiation.Br J Dermatol. 1985; 113: 65-76Crossref PubMed Scopus (64) Google Scholar;McKinlay and Diffey, 1987McKinlay A.F. Diffey B.L. A reference action spectrum for ultraviolet induced erythema in human skin.Cie J. 1987; 6: 17-22Google Scholar) is quite similar in trend to the action spectra for TT in the basal layers. Direct evidence that DNA is a chromophore for erythema has been obtained from animal studies (Ley, 1985Ley R.D. Photoreactivation of UV-induced pyrimidine dimers and erythema in the marsupialMonodelphis domestica.Proc Natl Acad Sci USA. 1985; 82: 2409-2411Crossref PubMed Scopus (97) Google Scholar). The overall similarity of the TT and erythema action spectra in our studies, especially in the 300–340 nm range, provide good circumstantial evidence that epidermal DNA is a chromophore for human erythema; however, it is highly improbable that there are different chromophores for different degrees of erythema induced by the same wavelength as might be suggested by spectral comparisons at 280–290 nm. A more likely explanation is that the precise location of the chromophore within the epidermis is the determining factor, but our data cannot exclude a role for DNA damage to dermal cells, e.g., endothelial cells, especially at wavelengths of 300 nm and higher. We speculate that a UVC and a shorter UVB wavelength MED is due to DNA damage in the mid to superficial epidermis. This results in the release of mediators, e.g., cytokines, which diffuse into the epidermis and activate the erythema response; however, a more intense erythema by these wavelengths may be due to DNA damage to deeper epidermal layers. An erythema action spectrum byAnders et al., 1995Anders A. Altheide H.-J. Knalmann M. Knälmann M. Tronnier H. Action spectrum for erythema in humans investigated with dye lasers.Photochem Photobiol. 1995; 61: 200-205Crossref PubMed Scopus (78) Google Scholar provides evidence for a distinct UVA absorbing chromophore with a peak at about 360 nm. Our data show evidence of a discrepancy between the erythema and TT action spectra between 340 and 360 nm, also providing some evidence for a chromophore other than DNA in the UVAI (340–400 nm) part of the spectrum. We speculate that at about 360 nm, erythema may result from absorption by at least two chromophores, one of which is DNA and the other unknown. We believe that we can exclude stratum corneum urocanic acid, which undergoes trans to cis isomerization after UVR exposure, as a possible chromophore for erythema in the UVB and UVA range. The action spectrum for urocanic acid photoisomerization in human skin is flat from about 280–310 nm, with activity falling off by less than an order of magnitude at 340 nm (Gibbs et al., 1997Gibbs N.K. McLoone P.R. Simics E. An action spectrum for urocanic acid isomerisation in human skin.In Vivo. Photochem Photobiol. 1997; 65S: 103S-104SGoogle Scholar).Figure 2 shows that 340 nm is three orders of magnitude less effective than 300 nm at erythema induction. As we have recently shown that the TT dose–response curves (expressed in individual MED units) for basal keratinocytes and melanocytes in skin types I/II are virtually identical at 300, 320, 340, and 360 nm (Young et al., 1998Young A.R. Potten C.S. Nikaido O. Parsons P.G. Boenders J. Ramsden J.M. Chadwick C.A. Human melanocytes and keratinocytes exposed to UVB or UVAin vivo show comparable levels of thymine dimers.J Invest Dermatol. 1998; 111: 936-940Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), it is reasonable to conclude that the TT action spectra for both cell types are the same. Thus, our data also indirectly support the hypothesis that DNA, a chromophore for melanogenesis (Gilchrest et al., 1996Gilchrest B.A. Park H.-Y. Eller M.S. Yaar M. Mechanisms of ultraviolet light-induced pigmentation.Photochem Photobiol. 1996; 63: 1-10Crossref PubMed Scopus (299) Google Scholar) as the human action spectra for the 24 h MED, and melanogenesis are the same in the 300–340 nm region (Parrish et al., 1982Parrish J.A. Jaenicke K.F. Anderson R.R. Erythema and melanogenesis action spectra of normal human skin.Photochem Photobiol. 1982; 36: 187-191Crossref PubMed Scopus (366) Google Scholar). The MED is the most widely used endpoint in clinical and experimental skin photobiology. It is also the endpoint in the determination of the sun protection factors of sunscreens. The role of sunscreens in the prevention of nonerythema endpoints, such as skin cancer, is of considerable topical interest (McGregor and Young, 1996McGregor J.M. Young A.R. Sunscreens, suntans, and skin cancer.Br Med J. 1996; 312: 1621-1622Crossref PubMed Scopus (34) Google Scholar). Our data suggest that human erythema is a good spectral surrogate for the UVB and UVAII (320–340 nm) component of sunlight-induced epidermal DNA damage to basal keratinocytes and melanocytes, which is an important factor in skin cancer. This conclusion, however, is not supported by a mouse study in which a UVA/UVB sunscreen was more effective than a UVB sunscreen, with comparable sun protection factors, at protecting from solar simulated radiation-induced CPD (Ley and Fourtanier, 1997Ley R.D. Fourtanier A. Sunscreen protection against ultraviolet radiation-induced pyrimidine dimers in mouse epidermal DNA.Photochem Photobiol. 1997; 65: 1007-1011Crossref PubMed Scopus (39) Google Scholar). In any case, it must be stressed that suberythemal doses of solar simulated radiation induce epidermal TT (Young et al., 1996Young A.R. Chadwick C.A. Harrison G.I. Hawk J.L.M. Nikaido O. Potten S. Thein situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II.J Invest Dermatol. 1996; 106: 307-1313Google Scholar), and data from this study, using monochromatic sources, confirm this observation. Thus the prevention of erythema alone by sunscreens cannot guarantee the prevention of epidermal DNA photodamage. We are grateful to the UK Government's Department of Health, the UK National Radiological Protection Board (NRPB), and Cancer Research Campaign UK for their generous support of this study. We thank Mrs. Jacqui Nagel for her excellent technical and administrative assistance with these studies. We are grateful to Dr. John Sutherland and Dr. Tsukasa Matsunaga for generously providing the raw data for the DNA absorption and TT in vitro action spectra, respectivelyFigure 7. We also thank Drs. Grant Bellany, Sabina Pfeifer, Charlotte Proby, Jane McGregor, and Sriramulu Tharakarum for taking the biopsies and John Sheehan for preparing the figures and references. Finally, we express our gratitude to all the volunteers for their participation in this study.